Fura 2 acetoxymethyl ester

All cell culture reagents were obtained from Invitrogen (Gaithersburg, MD, USA). Amaxa transfection kit (Kit V) was obtained from Lonza (Gaithersburg, MD, USA). Q5 Site-Directed Mutagenesis Kit was from New England BioLabs (Ipswich, MA). Substance P (SP) was from AnaSpec (Fremont, CA, USA). Pertussis toxin (PTx) was from List Biological Laboratories (Campbell, CA, USA). YM-254890 was from Wako Chemicals (Richmond, VA, USA) and p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) was from Sigma-Aldrich (St. Louis, MO, USA). Fura-2 acetoxymethyl ester was from Abcam (Cambridge, MA, USA). PE-conjugated anti-MRGPRX2 antibody was from BioLegend (San Diego, CA, USA). MRGPRX2 plasmid encoding hemagglutinin (HA)-tagged human MRGPRX2 in pReceiver-MO6 vector was obtained from GeneCopoeia (Rockville, MD, USA).

The cells (50,000–60,000 cells/well) were plated in 96-well plates and TRPM7 expression was induced 3 h post-plating by tetracycline in HEK-M7. The culture medium was completely removed after 18 h post-induction and replaced with fura-2 loading-buffer: 2 mM fura-2-acetoxymethyl ester (Abcam, Cambridge, UK) in ECS. Following incubation (60 min at 37 °C), the loading buffer was removed, and the cells were washed once with ECS before the addition of fresh ECS as the assay buffer. The plates were then transferred to a pre-warmed (37 °C) fluorescence plate reader ((PerkinElmer, Waltham, MA, USA) Victor X3). In the quench assay, cells were incubated with test substances or vehicles for 5 min after loading. Vehicle-receiving induced HEK-M7 cells served as a positive control for the activation of a TRPM7-mediated Mn²⁺-influx, while wells containing vehicle-receiving WT-HEK served as negative controls to define the nonspecific flux of Mn²⁺. The Ca²⁺-independent fluorescence (excitation 360 nm; emission 510 nm) of fura-2 was monitored following the addition of 10 mM MnCl₂. The method was tested by Waix A on HEK-M7 (Figure S4) and the dose–response curve was similar to the result reported in [30 (link)].

All cell culture and Lipofectamine 2000 transfection reagents were obtained from Invitrogen (Gaithersburg, MD, USA). Amaxa Nucleofector kit (Kit V) was purchased from Lonza (Gaithersburg, MD, USA). Phycoerythrin (PE)-conjugated (Cat.#359004) and purified unconjugated anti-MRGPRX2 antibodies (Cat.#359002) were from BioLegend (San Diego, CA, USA). Donkey anti-mouse Alexa Fluor 488 (Cat.#A21202) and 647 (Cat.#A31571) conjugated IgG secondary antibodies were from Invitrogen (Gaithersburg, MD, USA). p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) was from Sigma-Aldrich (St. Louis, MO, USA). Fura-2 acetoxymethyl ester was from Abcam (Cambridge, MA, USA). SP was purchased from AnaSpec (Fremont, CA, USA). Pertussis toxin (PTx) was from List Biological Laboratories (Campbell, CA, USA). Plasmid encoding hemagglutinin (HA)-tagged human MRGPRX2 in pReceiver-MO6 vector was obtained from GeneCopoeia (Rockville, MD, USA). MRGPRX2 Y279A mutant in HA-tagged plasmid was reported previously [28 (link)]. MRGPRX2-Tango plasmid (Addgene no. 66440) was a gift from Dr. Bryan Roth. MRGPRX2 Y279A mutant in Tango plasmid was generated by Penn Genomics Analysis Core (Philadelphia, PA, USA).